Accurate DNA replication is essential for normal development and preventing diseases such as cancer. The importance of the E3 ubiquitin ligases in coordinating myriad processes to ensure a single, precise round of replication, even under unfavorable conditions (replication stress), is well known. However, the mechanisms that regulate the ordered activities of these ligases are less clear. Dissecting these mechanisms is critical not only for understanding the pathophysiological alterations of this fundamental process, but also for exploiting these changes to develop novel therapeutic strategies. Recently, we showed that the deubiquitinating enzyme (DUB) USP37 binds and antagonizes the E3 ligase APCCdh1 to facilitate timely replication initiation. Further, proteomic analysis identified multipl APCCdh1 substrates and PCNA-associated factors in the USP37- interactome. USP37 is implicated in development and oncogenesis. However, its biological function(s) are nearly unknown. Our data thus far signify that USP37 is central to the replication process. Continuing this novel line of research, we have found that, first, sequential targeting of substrates by USP37 controls replication checkpoint activity. In late G1/early S, USP37 promotes the activation of the checkpoint. In late S/G2, USP37 facilitates checkpoint recovery and cell cycle resumption. Our data support a model whereby phosphorylation of USP37 in S-phase (likely by ATR) alters its substrate specificity, allowing the accumulation of key checkpoint proteins and increased checkpoint activity. Second, during S-phase we found that USP37 is recruited to chromatin and interacts with the molecular scaffold PCNA. Our data suggest that USP37 promotes genomic stability by regulating PCNA interactions to prevent spurious recruitment of replication factors. Consistent with a central role in replication, USP37-depleted cells had diminished survival on exposure to relatively inert replication stress. In this application we propose to elucidate the USP37-dependent mechanisms underlying control of replication checkpoint activity and regulation of PCNA interactions; these mechanisms will then serve as working models of USP37-substrate interactions for identifying and characterizing additional USP37 substrates and functions. Our central hypothesis is that regulation of USP37 substrate selection during the cell cycle promotes faithful genome replication and tolerance of replication stress. To test this hypothesis we will: 1. Determine that regulated USP37 substrate selection controls the replication checkpoint. 2. Characterize the impact of the USP37-PCNA interaction on the tolerance of replication stress 3. Investigate the USP37-regulated proteome. The results of these studies will provide significant new insight into how cells maintain genomic integrity. Ultimately, the project lays the foundation for dissecting the role of USP37 in embryonic development and the function of increased USP37 expression in cancer, which may ultimately provide the rationale for pursuing USP37 inhibition as a potent means of sensitizing cells to replication stress.